Nonalcoholic fatty liver disease (NAFLD) is marked by accumulation of fat in liver cells with accompanying inflammation and variable degrees of cell injury and fibrosis. When cell injury and fibrosis are present, the disease has a potential to progress and is referred to as nonalcoholic steatohepatitis (NASH). The etiology of NASH is not clear, but most patients are overweight or obese and have either insulin resistance or frank diabetes. Because of this association with obesity and diabetes, therapies for NASH have focused upon weight loss and improvement in insulin resistance. Starting in 2002, we conducted a series of clinical research studies in NASH. An initial study focused on the thiazolidinediones (TZDs), insulin sensitizing agents that are used widely in diabetes. In a pilot study, 22 non-diabetic patients with NASH were treated with pioglitazone (30 mg daily) and underwent extensive testing for metabolic status, body composition and liver disease (including liver biopsy) before and at the end of 48 weeks of therapy. Two-thirds of patients improved on therapy as judged by strict histological criteria. Improvements were accompanied by a marked decrease in hepatic fat despite an overall increase in body weight and total body fat. Thus, the effects of TZDs in NASH appeared to be due to the redirection of fat storage from the liver and central sites to the periphery. The histological improvements in the liver were not just in amount of fat (steatosis), but more strikingly in cell injury, inflammation and fibrosis. In follow up of this study, samples from patients were tested for a battery of cytokines and adipokines. Histological improvements correlated most clearly with changes in adiponectin, an adipokine that improves insulin signaling and induces maturation of adipocytes. When pioglitazone was stopped, the serum biochemical and histological features of NASH were reversed, histological scores returning to baseline by a year after discontinuation of pioglitazone. Importantly, the weight gain that occurred during pioglitazone therapy was not reversed; so that patients who received a one-year course of pioglitazone no longer had the histological benefit but were considerably heavier than before they were treated. These finds indicate that long-term improvement in NASH would require long-term therapy with a TZD and that the weight gain that often accompanies TZD therapy is likely to ultimately reverse any benefit. We completed a prospective, open-labelled study of metformin as therapy for NASH. A total of 28 patients with NASH were enrolled. The design of the study was similar to that for pioglitazone, in that patients underwent extensive evaluation of body composition, metabolic status, insulin sensitivity and liver disease (including liver biopsy) before and at the end of a 48 week course of metformin (2000 mg daily). The primary endpoint was histologic improvement. Of 28 patients enrolled, 26 (13 females; average age 44 years) completed 48 weeks of treatment and underwent repeat metabolic studies, imaging and liver biopsy. Thirty percent achieved a histologic response. Most patients lost weight, the average being 6 kg. There was a marked association between weight loss and improvements in NASH activity index and ALT levels (both, p <0.01). Insulin sensitivity also improved, but the degree of change did not correlate with histologic improvement. Thus, metformin leads to improvements in liver histology and ALT levels in 30% of patients with NASH probably by its effects in causing weight loss. Genome wide association (GWA) studies identified single nucleotide polymorphisms (SNPs) that are associated with increased hepatic fat or elevated liver enzymes, presumably reflecting nonalcoholic fatty liver disease (NAFLD). We initiated a study to investigate whether these SNPs are associated with histological severity in a large cohort of NAFLD patients. 1117 (894 adults/223 children) individuals enrolled in NASH-Clinical Research Network and National Institutes of Health Clinical Center studies with histologically-confirmed NAFLD were genotyped for six SNPs that are associated with hepatic fat or liver enzymes in GWA studies. In adults, 3 SNPs on chromosome 22 showed associations with histological parameters of nonalcoholic steatohepatitis (NASH). After adjustment for age, gender, diabetes and alcohol consumption, the minor allele of rs738409C/G, a nonsynonymous coding SNP in the PNPLA3 (adiponutrin) gene encoding an I148M change, was associated with steatosis (p=0.03), portal inflammation (p=2.5x10-4), lobular inflammation (p=0.005), Mallory-Denk bodies (p=0.015), NAFLD activity score (NAS, p=0.004) and fibrosis (p=7.7x10-6). Two other SNPs in the SAMM50-PNPLA3 cluster demonstrated similar associations. Three SNPs on chromosome 10 in the CPN1-ERLIN1-CHUK region were independently associated with fibrosis (p=0.010). In children, no SNP was associated with histological severity. However, the rs738409 G allele was associated with younger age at the time of biopsy in multivariate analysis (p=0.045). In this large cohort of histologically-proven NAFLD, we confirm the association of the rs738409G allele with steatosis and describe its association with histological severity. In pediatric patients, the high-risk rs738409G allele is associated with an earlier presentation of disease. We also describe a hitherto unknown association between SNPs at a chromosome 10 locus and the severity of NASH fibrosis. In a subsequent project we genotyped adult Caucasians from the same NAFLD cohort for another group of SNPs that are associated with liver enzyme activity. We demonstrated associations of SNPs near or in the genes for hydroxysteroid (17&#946;) dehydrogenase 13 (HSD17B13), RAR-related orphan receptor &#945; (RORA) and protein phosphatase 1, regulatory subunit 3B (PPP1R3B) with histological features of NAFLD, after adjustment for age, gender and BMI. In-depth genotyping near RORA, a nuclear receptor involved in control of circadian rhythm and metabolic functions, showed that SNPs that are associated with NAFLD are located in the putative promoter region of 2 of the 4 splice variants (variants 2 and 3) as opposed to SNPs upstream of other variants, suggesting that alternation in the relative expression of the different isoforms affects fat accumulation in the liver. HSD17B13 is predominantly expressed in the liver and colocalizes with lipid droplets, but its substrate and physiological roles are unknown. In-depth genotyping of the gene region demonstrated associations of coding SNPs in the gene with NAFLD, confirming a possible role for this enzyme in the pathogenesis of NASH. To better understand the roles of RORA and HSD17B13 in NAFLD and normal physiology, we studied their effect on fat accumulation in adipocytes and hepatocytes in culture. We found that siRNA knock-down of the expression of either RORA or HSD17B13, did not affect the degree of hepatic lipid accumulation under standard conditions. However, when cells were overloaded with nutrients (high glucose, high-fatty acids medium), the amount of intracellular fat significantly decreased with knock-down, predominantly through a decrease in the average size of the lipid droplets. A similar effect on adipocytes was seen by RORA, but not HSD17B13 knock-down. Future studies in NASH will be directed at other means of improving this liver disease. Possible therapies that are being considered include use of Vitamin E. We are also develop metabolic and physiologic studies of patients with NAFLD and/or metabolic syndrome and continue to study the functional significance of genetic variants and their role in disease pathophysiology and response to treatment.